CONCEPT DEVELOPMENT #4
DNA EXTRACTION
Day 1
1. Obtain individual E.coli colonies from a culture plate provided by your teacher. Use a sterile inoculating loop and sterile technique to suspend a 2-3 mm mass of E.coli in a 15 ml culture tube containing 4 ml of Luria broth.Label this Tube A.
2. Obtain another culture tube containing 4 ml of Luria broth without adding any E.coli.
Label this Tube B.
Incubate both tubes overnight at 37C or over three days at room temperature with frequent shaking.
Day 2
1.Add to both labeled tubes 3 ml of a 50% solution of dishwashing detergent in water. Shake each tube to assure complete mixing.2. Your teacher will provide a 65-75C water bath. Place each tube into the water bath for 15 minutes.
Note: Maintain water bath temperature accurately. >60C is needed to destroy enzymes, but temperatures >80C will denature (break apart) DNA.
3. Use a dropper to carefully pour 3 ml of 95% ethanol on top of the suspension of dissolved cell contents in each tube. (The alcohol should float in a layer on top). Push a glass rod through the alcohol into the suspension, stir and turn. The rod carries a little alcohol into the suspension, and makes DNA come out of solution onto the rod. Keep moving the rod through the alcohol into the cell suspension; each time a little more DNA should appear. Do not totally mix the two layers.